1. Field of Invention
The invention relates to a baculovirus aerosol infection method and, in particular, to an insect larva aerosol infection method that can be used to produce recombinant proteins and baculovirus bio-insecticides.
2. Related Art
Baculoviruses, among other insect viruses, are regarded as safe and as one of the bio-insecticides with the promising potential. They are DNA viruses with 90 to 160 kb genome size. Apart from the fact that they are effective bio-insecticides to replace chemical insecticides, gene recombination can enable the baculoviruses to carry foreign genes as a means to produce recombinant proteins in insect cells or in insect larvae. The baculovirus expression vector system (BEVS) is an expression vector system that uses insect cells or insect larvae as the host to produce recombinant proteins. There have been hundreds of proteins being successfully produced by the baculovirus expression vector systems since the year of 1983 when Smith et al. developed the baculovirus expression vector systems. Furthermore, recent research studies have found that the baculoviruses also have the potential to serve as the carriers of human gene therapy.
In comparison with mammal cells, the cell splitting time of insect cells is shorter and the nutrition requirement is more economical. The cost is far less than the expression vector systems of mammal cells. With the advantages of being easy to culture, having a shorter production time, and the fact that the baculoviruses have the virus property of exclusive infection, the baculovirus expression vector systems have become one of the most valuable biotechnologies tool. The baculovirus expression vector systems can be divided into the production procedures of cells and insect larvae. The baculovirus production procedure of cells involve such factors as insect cells, viruses, culture media, and culturing procedures. Its production is more complicated than the insect production procedure. The culture media have higher prices that increase the total cost. There is also a yield scale up problem as the mass culturing of cells is not easy.
The production procedure for recombinant proteins or virus based insecticide in insect larvae is performed by using viruses to infect insect larvae. In addition to the lower cost, this method also has the advantage that the yield of insects is far larger than that of cells. Therefore, it has been thought to be the best means for mass production. Generally, there are two major ways to infect insect larvae with baculoviruses: the infection among the individual insect larvae and the tissue cell infection of the insect itself. Insect larvae get the occlusion bodies (OB) through oral and then release budding virus (BV) particles in alkaline intestines, thereby infecting the intestine cells. The tissue cell infection of the insect itself is achieved by the fact that baculoviruses will produce budding form baculoviruses 12 hours after they enter intestine cells, infecting all tissue cells in the insect. In order to infect the insect larvae with baculovirus, one has to feed them with OV or inject BV one by one. The infection rate of feeding is hard to control. When the OV enters an insect, a lot of energy is wasted in generating OV proteins, reducing the yield of recombinant proteins. Therefore, most of current production technologies tend to infect insect larvae by injecting BV. However, such a method requires a lot of manpower and time. Consequently, how to effectively save manpower and at the same time to increase the baculovirus infection rate of the larvae has become an important subject in developing the technique of using BVES to produce recombinant proteins.
In 1994, Professor Volkman at UC Berkeley started to perform researches in the baculovirus infection paths. Her research results indicated that the insect tracheal system could be the cause that baculoviruses were able to rapidly infect all tissues in an insect larva. The Volkman research team found that 16 hours after feeding infection, 54% of the tracheoblasts was infected. That is the first non-midgut epithelial infected by baculoviruses. The tracheal systems of insect extend to the insect larvae surface, forming spiracles on the surface. Since the insect may be infected by the baculovirus through the spiracles, Volkman first anesthetized the insect larvae using carbon dioxide. At the same time, dry powders of insects containing OV and BV were sprayed over the insect larvae. According to the results published by Volkman in 1994, Trichoplusia nis, Heliothis virescenses, and Helicoverpa zeas can be infected via dry insect powder spray. However, the infection rate was neither high nor stable (8˜53%). It was also hard to determine how the aerosol baculovirus infection was spread among insects. Therefore, individual injection and feeding infection are still the mainstream in production. There are even sets of injection and culturing tools sold on the market.